It is the overarching goal of this project to develop host-directed inhibitors of myxovirus replication that are designed to transform the prevalent one-drug, one-bug approach of antiviral therapy to a one-drug, multiple-bugs paradigm. This pioneering approach aims to systemically address two major challenges frequently associated with viral therapeutics: pre-existing or rapidly developing resistance to the inhibitor, and a narrow indication spectrum of many antiviral drugs, which are aggravated by cost and low flexibility of the current pharmaceutical manufacturing process, viral evolution and the emergence of novel, highly pathogenic agents. We hypothesize that these obstacles can be overcome through therapeutic targeting of host cell pathways required for virus propagation, since individual viral mutations will likely not compensate for loss-of-function of a host pathway, and viruses of related families frequently depend on overlapping sets of host components for completion of their life cycle. Considering a higher potential for drug-induced side effects that is intrinsically associated with a host-directed approach, pathogens of the myxovirus families such as influenza virus, Nipah virus and respiratory syncytial virus that are predominantly associated with acute disease emerge as particularly suitable for this therapeutic strategy, since treatment time and thus host exposure to the drug remain limited. To meet future clinical needs of antiviral therapy, this project will build on the solid foundation provided by our established anti-myxovirus program and pursue the identification and molecular characterization of innovative small-molecule antivirals to establish a tangible foundation for the future development of confirmed therapeutic candidate status towards IND-filing status. Through a combination of automated screening and chemical elaboration, our program has previously identified a pool of small-molecule hits with broadened nanomolar to low-micromolar activity against influenza virus and members of the paramyxovirus family. In this supplement project that will adhere to a set of clearly defined milestones, a novel, innovative dual-myxovirus pathogen screening protocol will be implemented to further diversify the portfolio of existing myxovirus inhibitors, alleviating the risk of early stage failure (aim 1). In search of a subset of structurally diverse lead scaffolds that meet these milestones and warrant further development, active concentrations of newly discovered and existing hits will be determined for a diverse panel of cell lines and primary cells, the effect on exposed cells profiled, the point-of-arrest in virus replication assessed, and, for selected leads with the highest antiviral potency, the broader pathogen indication spectrum, molecular target, and key ADME properties determined (aim 2). It is the goal of these exercises to identify a lead scaffold and at least one structurally unrelated alternative suitable for synthetic development and pre-clinical assessment.